The present invention relates to use of a viral protein, SERP-1, its analogs and biologically active fragments thereof in combination with an immunosuppressant in the prevention and treatment of inflammatory and immune reactions associated with numerous injuries and disease conditions.
Inflammation is the body's reaction to injury and infection. Three major events are involved in inflammation: (1) increased blood supply to the injured or infected area; (2) increased capillary permeability enabled by retraction of endothelial cells; and (3) migration of leukocytes out of the capillaries and into the surrounding tissue (hereinafter referred to as cellular infiltration). Roitt et al., Immunology, Grower Medical Publishing, New York, 1989. Increased capillary permeability allows larger molecules to cross the endothelium that are not ordinarily capable of doing so, thereby allowing soluble mediators of immunity such as leukocytes to reach the injured or infected site. Leukocytes, primarily neutrophil polymorphs (also known as polymorphonuclear leukocytes, neutrophils or PMNs) and macrophages, migrate to the injured site by a process known as chemotaxis. At the site of inflammation, tissue damage and complement activation cause the release of chemotactic peptides such as C5a. Id. Complement activation products are also responsible for causing degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction and increases in vascular permeability. Mulligan et al. 1991 J. Immunol. 148:1479-1485.
The traversing of leukocytes from the bloodstream to extravascular sites of inflammation or immune reaction involves a complex but coordinated series of events. At the extravascular site of infection or tissue injury, signals are generated such as bacterial endotoxins, activated complement fragments or proinflammatory cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor (TNF) which activate leukocytes and/or endothelial cells and cause one or both of these cell types to become adhesive. Initially, cells become transiently adhesive (manifested by rolling) and later, such cells become firmly adhesive (manifested by sticking). Adherent leukocytes travel across the endothelial cell surface, diapedese between endothelial cells and migrate through the subendothelial matrix to the site of inflammation or immune reaction. Harlan et al., Adhesion-Its role in Inflammatory Disease, W.H. Freeman & Co., New York, 1992.
Although leukocyte traversal of vessel walls to extravascular tissue is necessary for host defense against foreign antigens and organisms, leukocyte-endothelial interactions often have deleterious consequences for the host. For example, during the process of adherence and transendothelial migration, leukocytes release oxidants, proteases and cytokines that directly damage endothelium or cause endothelial dysfunction. Once at the extravascular site, emigrated leukocytes further contribute to tissue damage by releasing a variety of inflammatory mediators.
Moreover, single leukocytes sticking within the capillary lumen or aggregation of leukocytes within larger vessels are responsible for microvascular occlusion and ischemia. Leukocyte-mediated vascular and tissue injury has been implicated in pathogenesis of a wide variety of clinical disorders such as acute and chronic allograft rejection, vasculitis, rheumatoid and other forms of inflammatory based arthritis, inflammatory skin diseases, adult respiratory distress syndrome, ischemia-reperfusion syndromes such as myocardial infarction, shock, stroke, organ transplantation, crush injury and limb replantation. Id.
Many other serious clinical conditions involve underlying inflammatory processes in humans. For example, multiple sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, circulating leukocytes infiltrate inflamed brain endothelium and damage myelin, with resultant impaired nerve conduction and paralysis. Yednock et al., 1992 Nature 366:63-66. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of tissue damage caused by self antigen directed antibodies. Auto-antibodies bound to antigens in various organs lead to complement-mediated and inflammatory cell mediated tissue damage. Theofilopoubs, A. N. 1992 Encyclopedia of Immunology, pp. 1414-1417.
Reperfusion injury is another condition associated with activation of the inflammatory system and enhanced leukocyte-endothelial cell (EC) adhesion. There is much evidence that adhesion-promoting molecules facilitate interactions between leukocytes and endothelial cells and play important roles in acute inflammatory reaction and accompanying tissue injury. For example, in acute lung injury caused by deposition of IgG immune complexes or after bolus i.v. infusion of cobra venom factor (CVF), neutrophil activation and the generation of toxic oxygen metabolites cause acute injury. Mulligan et al., 1992 J. Immunol. 150(6):2401-2405. Neutrophils (PMNs) are also known to mediate ischemia/reperfusion injury in skeletal and cardiac muscle, kidney and other tissues. Pemberton et al., 1993 J. Immunol. 150:5104-5113.
Infiltration of airways by inflammatory cells, particularly eosinophils, neutrophils and T lymphocytes are characteristic features of atopic or allergic asthma. Cotran et al., Pathological Basis of Disease, W. B. Saunders, Philadelphia, 1994. Cellular infiltration of the pancreas with resultant destruction of islet beta-cells is the underlying pathogenesis associated with insulin-dependent diabetes mellitis. Burkly et al. 1994 Diabetes 43: 529-534. Activation of inflammatory cells whose products cause tissue injury underlies the pathology of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Cotran et al., 1994. Neutrophils, eosinophils, mast calls; lymphocytes and macrophages contribute to the inflammatory response. Minute microabcesses of neutrophils in the upper epithelial layers of the dermis accompany the characteristic epidermal hyperplasia/thickening and scaling in psoriasis.
Various anti-inflammatory drugs are currently available for use in treating conditions involving underlying inflammatory processes. Their effectiveness however, is widely variable and there remains a significant clinical unmet need. This is especially true in the aforementioned diseases where available therapy is either of limited effectiveness or is accompanied by unwanted side effect profiles. Moreover, few clinical agents are available which directly inhibit cellular infiltration, a major underlying cause of tissue damage associated with inflammation. Thus, there is a need for a safe, effective clinical agent for preventing and ameliorating cellular infiltration and consequential pathologic conditions associated with inflammatory diseases, injuries and resultant perturbations of cytokine networks.
Serine proteinase inhibitors (hereinafter “serpins”) make up a superfamily of related proteins and have been found encoded by poxviruses from four different genera. Myxoma virus (MYX) is a leporipoxvirus that causes a virulent systemic infection, myxomatosis, in the European rabbit (Oryctolagus cuniculus). Significantly, myxomatosis is characterized by rapid disseminated infection, immunosuppression, and the presence of secondary, gram negative infections. A closely related leporipoxvirus, Shope fibroma virus (SFV), causes only a localized infection in the same host. SFV differs from the virulent myxoma virus in that it contains only a fragmented open reading frame (ORF) for a corresponding myxoma virus ORF designated SERP-1. A disruption of the SERP-1 ORF in myxoma virus or in the related malignant rabbit fibroma virus (MRV) results in attenuation of virus pathogenicity in O. cuniculus. Macen et al., 1993 Virology 195:348-363. Thus, SERP-1 has been generally implicated in the complex response to leporipoxyiral infection in its natural host, O. cuniculus. Although the absence of SERP-1 from myxoma virus apparently causes an increased immune response in rabbit, the mechanism by which SERP-1 acts as a virulence factor is unclear.
The SERP-1 polypeptide has been demonstrated to decrease intimal fatty cellular proliferation associated with restenosis in rabbits following balloon angioplasty. Lucas et al., 1994 J. Cell. Biochem. Suppl. 18A:286; Liu et al., 1993 Circulation 88:I-81.
It has been discovered in accordance with the present invention that the combination of SERP-1, SERP-1 analogs and biologically active fragments thereof and an immunosuppressant is capable of directly inhibiting the infiltration of tissue by inflammatory cells that are responsible for tissue damage in inflammatory diseases and disorders.